Processing
xGen™ Broad-Range RNA Library Preparation Kit
Comprehensive data from a wide range of RNA inputs
Compatible with a range of input types and quantities, a variety of indexing options for your research, as well as manual or automated systems. Assemble RNA-Seq libraries from 1st strand cDNA synthesis.
xGen NGS—made for broad range RNA library preparation.
Ordering
- Comprehensive data—for mapping rates, genes identified, and transcript coverage.
- Accommodates your samples with a single kit—consistent results from 10 ng to 1 µg total RNA or 100 pg to 100 ng mRNA input.
- Consistent libraries—minimal adapter dimers so adapter titration is not required for supported inputs.
- Save time, reduce costs—go from RNA to library in 4.5 hours with a wide variety of index options, ranging from up to 1536 UDI primer pairs (available with or without NormalaseTM), and up to 96 CDI primer pairs (with or without Normalase).
- Workflow—designed for easy automation.
Product details
The xGen Broad-Range RNA Library Prep Kit has a fast NGS transcriptomics research workflow with comprehensive transcript coverage and NGS data quality from a broad range of input quantities for Illumina® sequencing platforms. Leveraging proprietary AdaptaseTM technology, this RNA library prep kit enables stranded RNA library construction directly from 1st strand cDNA without the requirement for 2nd strand cDNA synthesis and degradation, or template-switching methods. The kit is compatible with manual and automated workflows, as well as upstream and downstream enrichment and depletion methods. It supports a variety of indexing options in research studies.
Figure 1. xGen Broad-Range Library Prep workflow: After RNA fragmentation, the reverse transcriptase step uses random primers to generate the first-strand cDNA. Next, Adaptase technology simultaneously performs tailing and ligation to incorporate the R2 Stubby Adapter to the 3’ ends of the cDNA molecules. The extension step produces a dsDNA duplex, while ligation adds the R1 Stubby Adapter to the 3’ ends of the primer-extended cDNA molecules. Finally, indexing PCR increases library yield, incorporates single or dual indexes, and results in full-length adapters at the ends of each molecule. In addition, bead-cleanup steps are needed after extension, ligation, and final indexing PCR steps.
The xGen Broad-Range RNA Library Prep Kit leverages an indexing PCR step to complete the fully indexed adapter sequences, using primers that anneal to the Stubby Adapters, to be fully compatible with Illumina sequencers. IDT supplies a variety of index configurations and strategies, including:
- Combinatorial dual indexing, up to 96 combinations and compatible with Normalase
- Unique dual indexing, up to 1536 unique dual indices and compatible with Normalase
Available in 16, 96, or 4x96-reaction kit sizes, the xGen Broad-Range RNA Library Prep Kit is offered at scales to support evaluation and adoption.
Automation
The xGen Broad-Range RNA Library Prep Kit protocol is readily automatable. A 10% overage volume of reagents is supplied to accommodate automation and additional reagent overage volume is available upon request.
Table 1. xGen Broad-Range RNA Library Prep Kit specifications.
| Feature | Specification | Benefit |
|---|---|---|
| Input quantity | 10 ng to 1 µg total RNA 100 pg to 100 ng mRNA | Supports a wide input range Consistent library output |
| RNA types supported | Poly(A)-enriched mRNA Ribo-depleted RNA Total RNA | Supports most RNA applications |
| Technology | Adaptase tailing and ligation of 1st strand cDNA | No 2nd strand cDNA No adapter titration Fewer dimers and duplicates detected in internal research studies (Figure 2) Maintains strandedness (≥97%) Higher mapping, transcript detection |
| Workflow time* | 4.5 hours | Less hands-on time |
| Kit reaction sizes | 16, 96, and 4x96 | Evaluation and adoption |
| Components provided | Fragmentation module RT module Library prep Polymerase | Complete solution for processing total, enriched, or depleted RNA from transcript to library |
| Indexing options | Combinatorial dual Unique dual Normalase compatible | Flexible for different sequencers, workflows, and applications |
| Multiplexing capability | Up to 1536 libraries | Save sequencing costs |
| Automation | Compatible with liquid handlers Custom packaging available | Supports high-throughput applications |
*Workflow time is based upon incubation times and expected times for hands-on-steps. Actual workflow time may vary depending on individual factors in your laboratory.
Product data
Reduced dimer formation means no adapter titration
Adaptase technology results in minimal adapter dimers, which means no need for adapter titration (Figure 2). Compared to leading RNA library kits, which can produce libraries with >10% adapter dimers and require adapter titration steps, the xGen Broad-Range RNA Library Prep Kit produces <1% adapter dimers and maintains ligation efficiency at all supported input levels.
Figure 2. Comparison of kits. Libraries were prepared using two different kits with the same quantity of input material (n = 1 per input quantity) and subjected to the same number of PCR cycles according to the xGen Broad-Range
protocol recommendations during library amplification. Representative library sizes, yields, and BioanalyzerTM (Agilent) traces illustrate typical libraries generated from the same input series of poly(A)-enriched Universal Human Reference
(UHR) RNA (Agilent 740000) or human brain mRNA (Takara 636102), when processed by either the xGen Broad-Range RNA Library Prep Kit (left panel) or an RNA library kit from an alternate supplier (right panel). The arrows indicate adapter dimers that
were generated during library preparation.
High mapping and transcript identification with lower duplication rates
Figure 3. Comparison of data obtained using different suppliers’ kits. Universal Human Reference (UHR) Total RNA (Agilent 740000) was enriched using the NEBNext® poly(A) mRNA Magnetic Isolation Module (NEB E7490), before being processed by the xGen Broad-Range RNA Library Prep Kit and other supplier kits K and N. For each kit evaluated, libraries were prepared at 10, 100 and 500 ng input, where n = 1 sample per input quantity. PCR amplification of each library was performed as follows: 10 cycles for 500 ng inputs, 12 cycles for 100 ng inputs, and 16 cycles for 10 ng inputs. Libraries were sequenced on a MiniSeqTM with 2x75 bp paired-end reads. Fastq files were downsampled to 2.2 million reads before analysis using STAR (mapping rate), RNASeqC (genes/transcripts detected), or Picard (duplication rate). The xGen Broad-Range RNA Library Prep Kit has high mapping percentage, detects more genes and transcripts, and has fewer duplicates.
Resources
*RUO—For research use only. Not for use in diagnostic procedures. Unless otherwise agreed to in writing, IDT does not intend for these products to be used in clinical applications and does not warrant their fitness or suitability for any clinical diagnostic use. Purchaser is solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations.
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